Dynamic Changes in C-Raf Phosphorylation and 14-3-3 Protein Binding in Response to Growth Factor Stimulation

Mirko Hekman, Stefan Wiese, Renate Metz, Stefan Albert, Jakob Troppmair, Joachim Nickel, Michael Sendtner, Ulf R. Rapp
2003 Journal of Biological Chemistry  
Phosphorylation events play a crucial role in Raf activation. Phosphorylation of serines 259 and 621 in C-Raf and serines 364 and 728 in B-Raf has been suggested to be critical for association with 14-3-3 proteins. To study the functional consequences of Raf phosphorylations at these positions, we developed and characterized phosphospecific antibodies directed against 14-3-3 binding epitopes: a monoclonal phosphospecific antibody (6B4) directed against pS621 and a polyclonal antibody specific
more » ... r B-Raf-pS364 epitope. Although 6B4 detected both C-and B-Raf in Western blots, it specifically recognizes the native form of C-Raf but not B-Raf. Contrary to B-Raf, a kinase-dead mutant of C-Raf was found to be only poorly phosphorylated in the Ser-621 position. Moreover, serine 259 to alanine mutation prevented the Ser-621 phosphorylation suggesting an interdependence between these two 14-3-3 binding domains. Direct C-Raf⅐14-3-3 binding studies with purified proteins combined with competition assays revealed that the 14-3-3 binding domain surrounding pS621 represents the high affinity binding site, whereas the pS259 epitope mediates lower affinity binding. Raf isozymes differ in their 14-3-3 association rates. The time course of endogenous C-Raf activation in mammalian cells by nerve growth factor (NGF) has been examined using both phosphospecific antibodies directed against 14-3-3 binding sites (6B4 and anti-pS259) as well as phosphospecific antibodies directed against the activation domain (anti-pS338 and anti-pY340/pY341). Time course of Ser-621 phosphorylation, in contrast to Ser-259 phosphorylation, exhibited unexpected pattern reaching maximal phosphorylation within 30 s of NGF stimulation. Phosphorylation of tyrosine 340/341 reached maximal levels subsequent to Ser-621 phosphorylation and was coincident with emergence of kinase activity. Taken together, we found substantial differences between C-Raf⅐14-3-3 binding epitopes pS259 and pS621 and visualized for the first time the sequence of the essential C-Raf phosphorylation events in mammalian cells in response to growth factor stimulation.
doi:10.1074/jbc.m309620200 pmid:14688280 fatcat:hzmcyvpkcjfgrhepubgn43lxzm